The goal of this proposal is to determine the mechanisms for loss of B cell tolerance to phospholipids and mechanisms for tissue damage by anti-phospholipid antibodies in NZW/BXSB F1 (W/B) mice that develop both proliferative glomerulonephritis and anti-phospholipid syndrome. Disease is accelerated in male W/B mice that carry the Yaa locus containing a reduplication of the TLR7 gene. We have shown that T cell help is required for initial activation of anti-phospholipid B cells and induction of high affinity IgG autoantibodies. We hypothesize that immune complex formation and opsonization of apoptotic particles by IgG anti-phospholipid antibodies results in delivery of nucleic acids to TLR containing endosomes. Engagement of TLRs induces both IFN&#945;that fosters plasma cell maturation and BAFF that decreases the stringency for B cell negative selection and enhances B cell survival and class switching. Although disease is initiated by T cells, we hypothesize that after class switched antibodies with high enough affinity to generate immune complexes accumulate, disease can then be propagated by T cell independent mechanisms that are prominent in males bearing excess TLR7. Because excess IFN&#945;production is a feature of SLE and IFN&#945;induces upregulation of TLR7, particularly in females, similar mechanisms may pertain in individuals with the IFN signature even though genetic TLR7 overexpression has not been found in humans with SLE. To address this hypothesis, we will use mice with an autoreactive transgenic heavy chain and examine B cell selection at sequential stages of the B cell developmental pathway. This strategy will allow us to examine the role of intrinsic alterations in B cell signaling, exogenous signals from the innate immune system and T cell help in the initial loss of tolerance to autoantigens and in the perpetuation of the anti-phospholipid response. Our data will also help determine why anti-phospholipid syndrome fails to respond to conventional immunosuppressive therapies and help suggest new strategies for treating this devastating syndrome. The second hypothesis that we wish to address is that pathogenic autoantibodies alone can cause the manifestations of the anti-phospholipid syndrome that are mediated by Fc receptor or complement dependent mechanisms but that the formation of thromboses requires other inflammatory mediators to activate the endothelium. We will test the hypothesis that one mechanism for this [unreadable]second hit[unreadable] is endothelial cell death mediated via the ceramide pathway. Our long term goal is to define the interactions of the crucial pathways that contribute to induction and propagation of the anti-phospholipid syndrome so as to best devise individualized therapies for patients with SLE.